Pseudocosmospora hypoxylicola (Nectriaceae), a new species from the French Alps Christian LECHAT Abstract: Pseudocosmospora hypoxylicola is described and illustrated based on material occurring on stro- Jacques FOURNIER mata of Hypoxylon fuscum on Alnus alnobetula in France. The placement of this fungus in the genus Pseudo- cosmospora and its status as a distinct species are based on the study of its sexual and asexual morphs, colour of colony in culture and phylogenetic comparison of ITS1-5.8S-ITS2 and LSU sequences with those of cos- mospora-like fungi having an acremonium-like asexual morph. Ascomycete.org, 12 (1) : 6–10 Keywords: Ascomycota, fungicolous Nectriaceae, Hypocreales, Hypoxylon, rdnA, taxonomy. Mise en ligne le 15/02/2020 10.25664/ART-0290 Résumé : Pseudocosmospora hypoxylicola est décrite et illustrée d’après du matériel récolté sur des stromas d’Hypoxylon fuscum sur Alnus alnobetula en France. Le placement de ce champignon dans le genre Pseudo- cosmospora et son statut d’espèce nouvelle reposent sur l’étude des stades sexué et asexué, la couleur de la colonie en culture et sur la comparaison phylogénétique des séquences ITS1-5.8S-ITS2 et LSU avec celles d’espèces de type cosmospora ayant une forme asexuée de type acremonium. Mots-clés : Adn ribosomal, Ascomycota, Hypocréales, Hypoxylon, Nectriaceae fongicoles, taxinomie. Introduction these were corrected. Phylogenetic analyses were performed online at www.phylogeny.lirmm.fr (dErEEPEr et al., 2008). maximum likeli- hood phylogenetic analyses were performed with PhymL 3.0 aLrT In the course of a field survey of Ascomycota in the French Alps (ZWIcKL, 2006), using the GTr + I + Γ model of evolution. branch sup- in August 2019, initiated by Ascomycete.org, a cosmospora-like port was assessed using the non-parametric version of the approx- species was collected on dead or effete stromata of Hypoxylon fus- imate likelihood-ratio test, implemented in PhymL SH-aLrT cum (Pers.) Fr. (Hypoxylaceae, Xylariales) on Alnus alnobetula (Ehrh.) (AnISImOVA & GAScUEL, 2006). nomenclature follows mycobank (Wes- K. Koch. This fungus was assigned to the Nectriaceae based on as- terdijk Fungal biodiversity Institute, Utrecht, The netherlands). comata changing colour in 3% KOH and lactic acid. The new species described herein is morphologically similar to the known cosmo- spora-like species occurring on dead or effete stromata of Diatry- Taxonomy paceae and Hypoxylon as recently revised and delimited by GräFEnHAn et al. (2011), HErrErA et al. (2013; 2015), LOmbArd et al. Pseudocosmospora hypoxylicola Lechat & J. Fourn., sp. nov. – Fig. (2015), and LEcHAT et al. (2019). Here we explain our morphological, 3 – mb 833853. cultural and phylogenetic results leading to the placement of this species in the genus Pseudocosmospora c.S. Herrera & P. chaverri Diagnosis: Ascomata bright red, occurring on dead or effete stro- and discuss the features supporting the segregation of P. hypoxyli- mata of Hypoxylon fuscum, non-stromatic, with verrucose as- cola from its relatives. cospores (7.5–)8–9.5(–10) × 4–4.5(–5) µm; greyish white colony and acremonium-like asexual morph in vitro. Holotype: FrAncE, Isère, Le bourg-d’Oisans, rb du Lac du Lauvitel, Material and methods 1505 m asl., on Hypoxylon fuscum on Alnus alnobetula, 29 Aug. 2019, leg. A. mombert, cLL19020 (LIP), ITS and LSU Genbank sequences: dry specimens were rehydrated and examined using the method mn886606 and mn885608. described by rOSSmAn et al. (1999). microscopic observations and Etymology: The epithet hypoxylicola refers to the host Hypoxylon measurements were made in water. The holotype is deposited in LIP and the Latin suffix -cola = inhabitor. herbarium (University of Lille, France). cultures of living specimens were made on PdA (Potato dextrose Agar) with 5 mg/l of strepto- Ascomata gregarious, solitary to most often clustered in small to mycin in Petri dishes 5 cm diam. incubated at 25°c. dnA extraction, large groups, basally slightly immersed in host tissues, non-stro- amplification, and sequencing were performed by ALVALAb matic, subglobose to widely obpyriform 180–240 µm high, 170– (Oviedo, Spain): total dnA was extracted from pure cultures blend- 220 µm wide (me = 220 × 200 µm, n = 20), laterally pinched when ing a portion of them using a micropestle in 600 µL cTAb buffer dry, bright red, turning purple in 3% KOH, yellow in lactic acid, with (cTAb 2%, nacl 1.4 m, EdTA pH 8.0 20 mm, Tris-Hcl pH 8.0 100 mm). a discoidal apex 80–110 µm diam, composed of clavate, thick-walled The resulting mixture was incubated for 15 min at 65ºc. A similar cells with orange wall, with a minute acute papilla 10 µm high, volume of chloroform:isoamylalcohol (24:1) was added and carefully 20 µm diam., composed of cylindrical to slightly clavate cells with mixed with the samples until their emulsion. It was then centrifuged pale yellow wall. Ascomatal surface composed of cells of unde- for 10 min at 13.000 g, and the dnA in the supernatant was precip- fined shape forming textura epidermoidea. Lateral ascomatal wall itated with a volume of isopropanol. After a new centrifugation of in vertical section 20–30(–35) µm thick, of two regions: outer region 15 min at the same speed, the pellet was washed in 70% cold 15–20(–25) µm thick, composed of subglobose to ellipsoidal, thick- ethanol, centrifuged again for 2 min and dried. It was finally resus- walled cells, 6–12 × 5–8 µm, with orange wall 2–3 µm thick; inner pended in 200 µL ddH2O. Pcr amplification was performed with the region 10–15 µm thick, composed of ellipsoidal, elongate, hyaline primers ITS1F and ITS4 (WHITE et al., 1990; GArdES & brUnS, 1993) for cells 5–14 × 4–5 µm. Asci unitunicate, cylindrical, short-stipitate, 70– ITS, while Lr0r and Lr5 (VILGALyS & HESTEr, 1990) were used to am- 75 × 6–7 µm, apex flat to rounded with a ring-like apical thickening, plify the 28S nLSU region. Pcr reactions were performed under a containing 8 uniseriate ascospores, becoming multiseriate when program consisting of a hot start at 95ºc for 5 min, followed by 35 mature; evanescent, narrowly moniliform paraphyses 2–2.5 µm cycles at 94ºc, 54ºc and 72ºc (45, 30 and 45 s respectively) and a diam, interspersed between asci. Ascospores ellipsoidal, (7.5–)8– final 72ºc step 10 min. Pcr products were checked in 1% agarose 10(–10.5) × 4–4.7(–5) µm (me = 9.5 × 4.5 µm, n = 40), equally 1-sep- gels, and positive reactions were sequenced with primer ITS4. chro- tate, slightly constricted at septum, verrucose, hyaline, becoming matograms were checked searching for putative reading errors, and pale yellowish brown when mature. 6 Fig. 1 – maximum likelihood phylogeny (-lnL = 1550.92269) of Pseudocosmospora spp. inferred by PhymL 3.0, model HKy85 from a 610 bp matrix of ITS sequences, rooted with Clonostachys rosea. Fig. 2 – maximum likelihood phylogeny (-lnL = 1765.86433) of Pseudocosmospora spp. inferred by PhymL 3.0, model HKy85 from a 825 bp matrix, based on LSU sequences, rooted with Clonostachys rosea. Ascomycete.org 7 Fig. 3 – a-h: Pseudocosmospora hypoxylicola (cLL19020 Holotype); a-b: Ascomata in natural environment; c: close-up of an ascoma in side view; d: Vertical section through lateral ascomatal wall; e: Asci and ascospores; f: culture at two weeks (Petri dish 5 cm diam); g: conidiophore, phialides and conidia from culture; h: conidia from culture. All microscopic illustrations in water. Scale bars: a-b = 200 µm; c = 50 µm; d-e = 10 µm; g-h = 5 µm. 8 Ascomycete.org Asexual morph in nature not observed. cosmospora-like fungus was never encountered on stromata col- Cultural characteristics: colony 0.5–1.5 cm diam after two weeks, lected in lowlands, mainly on Alnus glutinosa (L.) Gaertn., Carpinus aerial mycelium floccose, pale greyish in centre, white and sporulat- betulus L. and Corylus avellana L. One could have expected that such ing at margin, producing an acremonium-like asexual morph and a fairly conspicuous mycoparasite would have not remained over- diffusing a pale grey coloration in medium immediately around looked for long, which implies two different interpretations until colony. conidiophores simple, branched to verticillate, 60–150 µm more collections of P. hypoxylicola become available. long, 3–4 µm diam, flexuous, smooth, arising from smooth, septate Hypoxylon fuscum is suspected to be a complex of closely related hyphae 3.5–4 μm diam; conidiogenous cells monophialidic, termi- species, occurring on several members of Betulaceae Gray and ex- nal, apically subulate, with a minutely flared collarette, 25–52 µm hibiting a wide range of ascospore dimensions. An attempt to long, 2–3 µm diam at base, 1.5–2 µm diam at apex. conidia ellip- demonstrate a statistical correlation between host and ascospore soidal, 5–8 × 3–5 µm, rounded at ends or rounded at tip and atten- size was inconclusive because of a large overlap of ascospore di- uated at base, smooth, with or without a visible abscission scar. mension between collections from various hosts. However, it was shown that ascospores of collections of H. fuscum on Alnus alnobe- Results and discussion tula [as A. viridis (chaix. dc.)] were larger than those on other hosts and significantly larger than those on Carpinus betulus (PETrInI et al., 1987). The peculiar ecology of Alnus alnobetula, ranging from sub- Fungicolous Nectriaceae formerly accommodated in Cosmospora alpine to alpine zone, suggests a possible speciation process within sensu rOSSmAn et al. (1999) were shown to be para/polyphyletic by the H. fuscum complex, which might involve a specific mycoparasite. GräFEnHAn et al. (2011), who segregated Cosmospora sensu stricto If so, an active search on stromata of H. fuscum on A. alnobetula typified by C. coccinea rabenh. for species with acremonium-like should lead to the discovery of additional collections of P.